The organization of the vertebrate brain is established during gastrulation and early somitogenesis. Anterior-posterior molecular gradients posteriorize the neural plate, and position boundaries between major domains of the brain. The boundaries serve as regional organizing centers that pattern the brain and act in neuronal specification. The vertebrate isthmic organizer, or midbrain-hindbrain boundary, organizes the midbrain and hindbrain, and is required for development of the serotonergic and dopaminergic systems, major components in neurological disease. Long-term objective: to learn how isthmic development is initiated. Isthmic initiation fails in zebrafish spiel ohne grenzen (spg) mutants: midline, or ventral, expression of isthmic genes is absent; while lateral, or dorsal, is reduced; and no isthmus forms. spg mutants have lesions in pou2; which is expressed in the midbrain, anterior hindbrain, and midline during isthmic initiation; the first POU- domain transcription factor shown to act in isthmic initiation. Specific Aims: (i) Test whether the dorsal-ventral effect on isthmic gene expression in pou2 mutants stems from a role of pou2 in FGF-mediated vertical signaling required for neural regionalization. (ii) Find regulators of pou2, by analyzing genes coexpressed early with pou2, mutant epistasis analyses, and genetic mutagenesis. (iii) Assay candidate downstream target gene expression in wild-type vs. pou2 mutants to find genes regulated by pou2 in isthmic initiation. (iv) If pou2 targets are found, analyze whether pou2 activates or represses them. (V) Determine if maternal pou2 contributes to isthmus formation.